Low molecular weight acetylhyaluronate, skin-softening composition, method of manufacturing the same, and method of purifying the same

ABSTRACT

Low molecular weight acetylhyaluronate has the following characteristics: 
     intrinsic viscosity: 50 to 200 cm 3  /g 
     substitution degree of acetyl group: 2.6 to 3.6 (excluding N-acetyl group) 
     A skin-softening composition is mainly composed of this low molecular weight acetylhyaluronate. It can exhibit excellent skin-softening effect and moisturizing effect, while appropriately suppressing the thread-forming ability inherent in hyaluronic acid. A method of manufacturing acetylhyaluronate comprises the steps of suspending hyaluronic acid powder in an acetic anhydride solvent and then adding concentrated sulfuric acid thereto to effect acetylation. A method of purifying acetylhyaluronate comprises the steps of adding raw acetylhyaluronate to an aqueous acetone solution, adding and dissolving sodium lactate therein, and then adding highly concentrated acetone thereto to precipitate highly pure acetylhyaluronate. Acetylhyaluronate having highly purity can be obtained inexpensively.

FIELD OF THE INVENTION!

The present invention relates to a low molecular weightacetylhyaluronate, a skin-softening composition, a method ofmanufacturing the same, and a method of purifying the same. Inparticular, it relates to improvement in acetylhyaluronate in whichacetyl group is bonded to alcoholic hydroxyl group thereof with a highrate.

BACKGROUND OF THE INVENTION!

Hyaluronic acid is a high molecular weight material derived fromorganisms and has specific physical characteristics such as high viscousproperty, viscoelasticity, and thread-forming ability, while having highbiocompatibility. Accordingly, it is expected to be applied to variousfields.

In particular, it should be a notable physical characteristic thathyaluronic acid has a very high moisturizing effect. Accordingly, it isused as a humectant in various external preparations for skin. Further,it is expected to be used as a thickener in organic solvent systems,various emulsification stabilizers in oily bases, a coating enforcementfor liposomes, an embedded base for organisms, a capsule base, or thelike.

However, since hyaluronic acid has various kinds of specific physicalcharacteristics mentioned above, due to a part of its characteristics,it may deteriorate the usability of a product when a large amount of itis added thereto.

Also, since it is highly water-soluble, hyalucronic acid cannot be usedas a humectant in organic solvent systems or oily bases.

DISCLOSURE OF THE INVENTION!

In view of the foregoing problems of the prior art, the first object ofthe present invention is to provide inexpensive methods formanufacturing and purifying acetylhyaluronate which has various kinds ofspecific physical characteristics while maintaining the functionsinherent in hyaluronic acid.

The second object of the present invention is to provide low molecularweight acetylhyaluronate which can exhibit an excellent skin-softeningeffect while improving shortcomings in hyaluronic acid such asthread-forming ability.

As a result of diligent studies conducted by the inventors in order toattain the above-mentioned objects, it has been found thatacetylhyaluronate can be inexpensively manufactured when hyaluronic acidis reacted in the presence of acetic anhydride and concentrated sulfuricacid and that a specific kind of acetylhyaluronate having specificlimiting viscosity and substitutional number of acetyl group, namely,specific molecular weight and hydrophobic property, can be remarkablyimproved in terms of thread-forming ability and can exhibit a high levelof skin-softening effect. Thus, the present invention has beenaccomplished.

Namely, low molecular weight acetylhyaluronate in accordance with thepresent invention is characterized by the following characteristics:

intrinsic viscosity: 50 to 200 cm³ /g

substitution degree of acetyl group: 2.6 to 3.6 (excluding N-acetylgroup)

Here, in the present invention, the intrinsic viscosity refers to thatmeasured in phosphate buffer solution of pH 7.4 at 25° C.

The a skin-softening composition in accordance with the presentinvention is mainly composed of low molecular weight hyaluronic acid.

The method of manufacturing acetylhyaluronate in accordance with thepresent invention is characterized by comprising the steps of suspendinghyaluronic acid powder in an acetic anhydride solvent and then addingconcentrated sulfuric acid thereto to effect acetylation, therebyyielding acetylhyaluronate.

In the above-mentioned method, acetic acid may preferably be mixed intothe acetic anhydride solvent with a mixing ratio of acetic acid:aceticanhydride at 1:4 to 1:1. In this case, highly acetylhyaluronate having ahigh acetylation rate can be obtained.

Also, acetic acid may preferably be mixed into the acetic anhydridesolvent with a mixing ratio of acetic acid:acetic anhydride at 2:1 to4:1. In this case, acetylation advances mildly, whereby minute controlof the acetylation rate can be easily effected.

Preferably, an amount of concentrated sulfuric acid added is 2 to 7% byvolume with respect to the solvent.

The method of purifying acetylhyaluroate in accordance with the presentinvention is characterized by comprising the steps of adding rawacetylhyaluronate to an aqueous acetone solution, adding and dissolvingsodium lactate therein, and then adding highly concentrated acetonethereto to attain acetylhyaluronate having highly purity.

Preferably, an amount of sodium lactate added is 1 to 3% by weight withrespect to the aqueous acetone solution.

Also, preferably, the above-mentioned purifying method is applied to rawacetylhyaluronate obtained by the above-mentioned manufacturing method.

In the following, the constituents of the present invention will beexplained in further detail.

Low molecular weight acetylhyaluronate in accordance with the presentinvention has a structure shown in the following structural formula 1:##STR1##

When low molecular weight acetylhyaluronate in accordance with thepresent invention is used for a skin-softening composition, it isnecessary for the molecular weight thereof, as expressed by itsintrinsic viscosity, to be within the range of 50 to 200 cm³ /g asmentioned above. Here, the intrinsic viscosity is used since it isdifficult for the molecular weight of acetylhyaluronate to be directlyindicated.

When the intrinsic viscosity is 50 cm³ /g or less, it becomes harder toobtain the skin-softening effect resulting from acetylhyaluronate. Onthe other hand, when the intrinsic viscosity is 200 cm³ /g or more,unfavorable physical characteristics such as thread-forming abilitycannot be fully suppressed even by acetylation.

The degree of acetylation, expressed by the substitution degree ofacetyl group, is preferably not less than 2.6 but not greater than 3.6.Here, while hyaluronic acid has four alcoholic hydroxyl groups in itsrepeating unit as shown in the above-mentioned structural formula 1, thesubstitution degree of acetyl group indicates how many of these groupsare substituted by acetyl group.

When the substitution degree of acetyl group is less than 2.6, thehydrophobic characteristic tends to be insufficiently provided. When thesubstitution degree of acetyl group exceeds 3.6, on the other hand, thehydrophilic characteristic may be lowered or usability may bedeteriorated, for example.

Japanese Unexamined Patent Publication No. 3-143540, for example,discloses an emulsification stabilizer in which an acyl group such asacetyl group is introduced in the repeating unit of hyaluronic acid.However, this hyaluronic acid. However, this hyaluronic acid derivativehas a very low modification ratio with acyl group/N-acetyl group ratioof less than a few tenths. Namely, only one acyl group is introduced inevery several or several ten repeating units. Accordingly, effects ofthe present invention cannot be obtained thereby. Also, when used as anemulsifier, the function for emulsifier cannot be practically attainedunless highly oily acyl group such as palmitoyl group is introducedtherein. Further, since pyridine system is used, hyaluronic acid may beremarkably decomposed when its modification ratio is intended to beincreased, thereby losing the inherent function of hyaluronic acid.

Also, Japanese Unexamined Patent Publication Hei No. 6-9707 discloses amethod of manufacturing highly acetylhyaluronate. Though this method isexcellent in that a high acetylation ratio can be obtained under a mildcondition, expensive agents are used therein. Accordingly, there hasbeen a demand for easier and less expensive methods for manufacturingacetylhyaluronate. Also, this is rather directed to high molecularweight acetylhyaluronate and not suggest that low molecular weightacetylhyaluronate has a high skin-softening effect as in the case of thepresent invention.

Preferably, acetylhyaluronate in accordance with the present inventionis made in the following manner.

Namely, hyaluronic acid powder is suspended in an acetic anhydridesolvent and then concentrated sulfuric add is added thereto to effectacetylation.

At this time, acetic acid is preferably mixed into the acetic anhydridesolvent with a mixing ratio of acetic add:acetic anhydride at 1:4 to1:1. In this case, low molecular weight acetylhyaluronate having a highacetylation rate can be obtained.

Also, when the mixing ratio of acetic acid:acetic anhydride is 2:1 to4:1, the degree of acetyl group substitution can be finely controlled.

Preferably, an amount of concentrated sulfuric acid added is 2 to 7% byvolume with respect to the solvent.

On the other hand, acetylhyaluronate in accordance with the presentinvention can be purified when raw acetylhyaluronate is added to anaqueous acetone solution, sodium lactate is added and dissolved therein,and then acetone is added thereto, thereby yielding acetylhyaluronatehaving high purity.

Here, 1 to 3% by weight of sodium lactate with respect to the aqueousacetone solution is added, preferably.

In the present invention, "hyaluronic acid" refers to hyaluronic acidand its salts and may have various molecular weights.

In the method of manufacturing acetylhyaluronate in accordance with thepresent invention, upon enzymatic treatment using such as hyaluronidase,hyaluronic acid having a wide range of acetylation rate with a molecularweight from oligohyaluronic acid to 10,000 kd or more can be obtained.Also, the modification rate can be altered by changing the reaction timeof esterification.

In the method of purifying acetylhyaluronate in accordance with thepresent invention, sodium lactate is added in order to depositacetylhyaluronate due to its salting out effect. Namely, sodium lactateis dissolved beforehand, for example, in 80% aqueous acetone solutionwhich can dissolve acetylhyaluronate. When the acetone content isincreased until 92% by addition of acetone, acetylhyaluronate isdeposited as a gel-like precipitate. As a salt used for this salting outprocess, sodium acetate, tri-sodium citrate, sodium glutamate, sodiumchloride, sodium pyrrolidonecarbonate, sodium tartrate, glycine,magnesium sulfate, and potassium chloride have been studied. Whilesodium acetate has exhibited some salting out effect, no substantialeffects have been recognized in the others. On the other hand, sodiumlactate has exhibited quite favorable effects.

The purifying method in accordance with the present invention utilizesthe original solvent-solubility of acetylhyaluronate made by theabove-mentioned manufacturing method. Acetylhyaluronate having highpurity can be obtained with a favorable yield in particular when themanufacturing method and purifying method in accordance with the presentinvention are combined together.

With regard to the amount of sodium lactate added, while the recovery ofacetylhyaluronate can be improved when the amount of added sodiumlactate is increased, the subsequent step for removing sodium lactate byethanol may become difficult thereby. Accordingly, the minimum amount ofsodium lactate added was studied in order that the recovery ofacetylhyaluronate becomes high as well as the remaining rate of sodiumlactate is low by control of the amount of acetone added whenacetylhyaluronate was deposited as a gel-like precipitate upon increasein acetone concentration. As a result, it has been determined that anamount of sodium lactate is 1 to 3% by weight with respect to theaqueous acetone solution.

As explained in the foregoing, in the method of manufacturingacetylhyaluronate in accordance with the present invention,acetylhyaluronate can be manufactured inexpensively.

Also, in the method of purifying acetylhyaluronate in accordance withthe present invention, purifying of acetylhyaluronate can be efficientlyadvanced.

Thus obtained acetylhyaluronate may have remarkably different physicalcharacteristics according to its viscosity, modification ratio, and thelike. For example, when it has a high viscosity and a medium degree ofmodification, a stable gel may be formed upon addition of a small amountof an organic solvent thereto so as to be expected to apply as a basefor cosmetic or for drag delivery systems.

Also, when it has a high viscosity and a high acetylation or it has alow viscosity, it can be dissolved in an organic solvent having aconsiderable concentration, and it can be easily blended in emulsifiedlotion, for example. When it is blended in emulsified lotion or thelike, various effects such as improvement in smoothness during use canbe exhibited. Also, when its lipo-solubility is increased uponacetylation, its affinity to a stratum corneum whose surface is a lipidmembrane can be increased, thereby improving its biocompatibility.

Further, highly acetylhyaluronate in accordance with the presentinvention has an advantageous effect that thread-forming ability, whichis a shortcoming of hyaluronic acid as well, can be remarkably reducedwhen blended in cosmetics or the like.

In the a skin-softening composition of the present invention, inaddition to the above-mentioned constituents, various ingredients whichhave been conventionally blended in external preparations for skin canbe blended. Examples of such ingredients include oil contents such asliquid paraffin, squalane, lanolin derivatives, higher alcohols, variousester oils, silicone oil, polyalkyleneglycol polyether and othercarboxylic acids, oligoester compounds, and terpene type hydrocarbonoils; surface active agents; ultraviolet absorbents; ultravioletscattering agents; resins such as acrylic resin, silicone resin, andpolyvinylpyrrolidone; proteins or protein decomposition products such assoybean protein, gelatin, collagen, silk fibroin, and elastin;antiseptics such as ethyl paraben and butyl paraben; activating agentssuch as biotin and pantothenic acid derivatives; diluents such asethanol, isopropanol, tetrachlorodifluoroethane, and toluene; viscosityenhancing agents such as carboxyvinylpolymers; chelating agents;antioxidants; humectants; medicaments; perfumes; and coloring agents.

BRIEF DESCRIPTION OF THE DRAWINGS!

FIG. 1 is an explanatory chart showing the relationship between thereaction time of acetylation and the intrinsic viscosity when thesolvent ratio of acetic acid to acetic anhydride is changed in themethod of manufacturing acetylhyaluronate in accordance with the presentinvention;

FIG. 2 is an explanatory chart showing the relationship between thereaction time of acetylation and the substitution degree of acetyl groupwhen the solvent ratio of acetic acid to acetic anhydride is changed inthe method of manufacturing acetylhyaluronate in accordance with thepresent invention;

FIG. 3 is an explanatory chart showing the relationship between thereaction time of acetylation and the intrinsic viscosity when the amountof reaction catalyst (concentrated sulfuric acid) is changed in themethod of manufacturing acetylhyaluronate in accordance with the presentinvention;

FIG. 4 is an explanatory chart showing the relationship between thereaction time of acetylation and the substitution degree of acetyl groupwhen the amount of reaction catalyst (concentrated sulfuric acid) ischanged in the method of manufacturing acetylhyaluronate in accordancewith the present invention;

FIG. 5 is an explanatory chart showing the thread length inacetylhyaluronatein accordance with the present invention;

FIG. 6 is an explanatory chart showing the water evaporating rate inacetylhyaluronate in accordance with the present invention; and

FIG. 7 is an explanatory chart showing the stratum corneum softeningeffect of acetylhyaluronate in accordance with the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION!

In the following, the present invention will be explained with referenceto its examples. However, the present invention should not be limited tothe following examples.

MANUFACTURING EXAMPLE 1 Method of Manufacturing Acetylhyaluronate

Commercially available guaranteed acetic acid (20 ml) and aceticanhydride (80 ml) were introduced into a conical glass flask having acapacity of 300 ml and fine powder of Biohyalo-12 (hyaluronic acidhaving a molecular weight of about 1,200 kd, manufactured by ShiseidoCo., Ltd.) (6 g) was gradually added thereto while being stirred. Then,concentrated sulfuric acid (4 ml) was gradually added to the mixture andstirred for one hour at room temperature so as to effect acetylationreaction. The reaction solution became a viscous white solution. Whenthe degree of substitution of thus obtained acetylhyaluronate wasmeasured, it was found to be a tri-substituted product. The yieldcalculated on the basis thereof was 88.8%.

PREPARATION EXAMPLE 1 Method of Purifying Acetylhyaluronate

Into a glass beaker having a capacity of 3 L, pitied water (2 L) wasintroduced and the above-mentioned reaction solution was gradually addedthereto like a string. The resulting precipitate of acetylhyaluronatewas collected and washed twice similarly with purified water (2 L). Thisprecipitate was transferred to a glass beaker having a capacity of 1 Land then 80% (v/v) aqueous acetone solution (250 ml) and 50% aqueoussodium lactate solution (9 g) were added thereto, so as to completelydissolve the precipitate while being stirred. Subsequently, acetone (400ml) was gradually added to the solution, thereby depositing the gel-likeprecipitate of acetylhyaluronate again. After this precipitate had beencollected, it was subjected to two sets of washing with ethanol (100 ml)by using a homogenizer at 10,000 rpm for 10 minutes. Then, theprecipitate was collected by filtration under reduced pressure and driedunder vacuum, thereby yielding a white powder of acetylhyaluronate.

As a result, 6.5 g of acetylhyaluronate (yield of 82.5%) was obtained.While 0.2% of lactic add with respect to this product was remaining,neither acetic acid nor sulfuric acid was detected.

Also, this product was soluble in 90% ethanol.

Influence of Reaction Solvent Ratio

Next, the inventors studied the relationship between the reaction timeof acetylation and the intrinsic viscosity or substitution degree ofacetyl group of acetylhyaluronate generated, when the ratio of aceticacid to acetic anhydride is changed under basically the same conditionas that of the above-mentioned Manufacturing Example 1.

FIG. 1 shows the relationship between the reaction time of acetylationand the intrinsic viscosity and FIG. 2 shows that between the reactiontime of acetylation and the substitution degree of acetyl group.

As can be seen from these charts, the substitution degree of acetylgroup drastically increases within several hours or, in particular,within an hour after the reaction is started. On the other hand, theviscosity drastically decreases until about 5 to 10 hours after thereaction is started. Accordingly, preferably, the reaction time is keptat one hour or less in order to obtain acetylhyaluronate having a highviscosity, while it is extended to the range of 5 to 10 hours in orderto obtain acetylhyaluronate having a low viscosity.

With respect to the solvent ratio, on the other hand, the advance ofacetylation is improved when acetic acid is somewhat added thereto ascompared with the case where acetic anhydride is used alone. Inparticular, when the ratio of acetic acid:acetic anhydride is about 1:4,acetylation advances more effectively. Even when the ratio of aceticacid:acetic anhydride is about 1:1, acetylation advances and viscositydecreases to substantially the same extent as that in the case whereacetic anhydride is used alone.

Accordingly, the solvent ratio of acetic acid to acetic anhydride foreffectively advancing acetylation is preferably at 1:4 to 1:1 inparticular. In order to adjust the substitution degree of acetyl groupto a low level, on the other hand, the ratio of acetic acid:aceticanhydride is rather held at about 1:2 to 4:1, whereby the fluctuation inthe substitution degree of acetyl group becomes small over time and itsadjustment becomes easy.

Change in Reaction Catalyst Amount

While sulfuric acid becomes a catalyst for advancing acetylation ofhyaluronic acid, the amount of this reaction catalyst also influencesthe intrinsic viscosity and substitution degree of acetyl group inacetylhyaluronate generated thereby. Under the same condition as theabove-mentioned Manufacturing Example 1, the relationship between thereaction catalyst amount and the intrinsic viscosity (FIG. 3) and thatbetween the reaction catalyst amount and the substitution degree ofacetyl group (FIG. 4) were studied when the mount of sulfuric acid waschanged.

As can be seen from these charts, while the degree of acetyl groupsubstitution increases as the amount of sulfuric acid increases, theviscosity decreases substantially in proportion thereto.

With respect to thus obtained acetylhyaluronate, the followingusefulness tests were conducted. Namely, thread-forming ability test,moisture evaporation test, and skin-softening effect measurement testwere respectively conducted concerning its usability, moisturizingeffect, and skin-softening effect.

Thread-Forming Ability Test

In a thermo-hygrostat (at a temperature of 25° C. and a relativehumidity of 50%), 1 cm of a glass rod having a diameter of about 7 mmwas immersed in a sample solution with a concentration of 2% containedin a glass beaker having a capacity of 100 ml. Then, the thread lengthobtained when the beaker was descended at a velocity of 5 cm/min wasmeasured.

Water Evaporation Test

It was based on occlusion effect (moisture nonvolatility) measurementtest method.

Into filter paper (No. 2), 0.5 ml of a sample solution having aconcentration of 1% was uniformly infiltrated and dried overnight underreduced pressure at room temperature. Then, this filter paper wasattached to an opening portion of a plastic Petri dish containing 5 mlof water and, in a thermo-hygrostat (at a temperature of 25° C. and arelative humidity of 50%), its change in weight was measured over time.When being left for a certain time, the weight of the sample solutiondecreased in proportion to time. Accordingly, the weight of the testsample was plotted with reference to the time passed since it had beenleft. The gradient of its approximate line was defined as constant ofwater evaporating rate k (mg/min), from which the occlusion effect wasevaluated. Namely, the occlusion effect is higher as the value of k issmaller.

Skin-Softening Test

It was based on stratum corneum softening effect test method.

A keratin layer collected from the back of a guinea pig by a heating andtrypsin treatment process was cut into a slice of 2×30 mm. It was set ina continuous dynamic viscoelasticity measurement apparatus (manufacturedby Toyo Seiki Co., Ltd and Shiseido Co., Ltd) and its elastic moduluswas measured three times at every three minutes over time. The avengethereof was defined as the measured value of the elastic modulus beforeapplying the sample solution. Then, 2 μl of the sample solution wasapplied to the keratin layer and spread thereon with a width of 3 to 4mm. The measurement was similarly conducted for a period of two hours.Then, the stratum corneum softening effect was evaluated in terms of theratio of the elastic modulus after the application to that before theapplication. Namely, the stratum corneum softening effect is higher asthe ratio of elastic modulus is smaller.

Thread-Forming Ability Pretest

Thread-forming ability test was conducted as mentioned above withrespect to the following sample solutions:

Sample solution 1: 0.2% aqueous solution of sodium hyaluronate (HA)having a molecular weight of 1,200,000;

Sample solution 2: 0.2% aqueous solution of low molecular weight sodiumacetylhyaluronate having a intrinsic viscosity of 170 cm³ /g and asubstitution degree of acetyl group of 2.7;

Sample solution 3: 0.2% aqueous solution of low molecular weight sodiumacetylhyaluronate having a intrinsic viscosity of 160 cm³ /g and asubstitution degree of acetyl group of 2.8;

Sample solution 4: 0.2% aqueous solution of low molecular weight sodiumacetylhyaluronate having a intrinsic viscosity of 140 cm³ /g and asubstitution degree of acetyl group of 2.9;

Sample solution 5: 0.2% aqueous solution of low molecular weight sodiumacetylhyaluronate having a intrinsic viscosity of 120 cm³ /g and asubstitution degree of acetyl group of 3.0;

Sample solution 6: 0.2% aqueous solution of low molecular weight sodiumacetylhyaluronate having a intrinsic viscosity of 110 cm³ /g and asubstitution degree of acetyl group of 3.1; and

Sample solution 7: 0.2% aqueous solution of low molecular weight sodiumacetylhyaluronate having a intrinsic viscosity of 100 cm³ /g and asubstitution degree of acetyl group of 3.2.

The results thereof are shown in FIG. 5.

As shown m this chart, the aqueous sodium hyaluronate solution has athread length of about 2.0 cm. On the other hand, each of the aqueoussodium acetylated hyaluronate solutions has a thread length within about0.1 to 0.4 cm which is significantly shorter than that in the aqueoussodium hyaluronate solution. In view of these results, it can be seenthat the thread-forming ability has been clearly improved.

Based on these results, it has been decided that the thread-formingability would be evaluated by the thread length.

Water Evaporation Measurement Pretest

Occlusion effect measurement test was conducted as mentioned above withrespect to the following sample solutions:

Sample solution 1: ion-exchanged water;,

Sample solution 2: 0.2% aqueous solution of tow molecular weight sodiumacetylhyaluronate having a intrinsic viscosity of 200 cm³ /g and asubstitution degree of acetyl group of 2.6;

Sample solution 3: 02% aqueous solution of low molecular weight sodiumacetylhyaluronate having a intrinsic viscosity of 170 cm³ /g and asubstitution degree of acetyl group of 2.7;

Sample solution 4: 0.2% aqueous solution of low molecular weight sodiumacetylhyaluronate having a intrinsic viscosity of 160 cm³ /g and asubstitution degree of acetyl group of 2.8;

Sample solution 5: 0.2% aqueous solution of low molecular weight sodiumacetylhyaluronate having a intrinsic viscosity of 120 cm³ /g and asubstitution degree of acetyl group of 3.0;

Sample solution 6: 0.2% aqueous solution of low molecular weight sodiumacetylhyaluronate having a intrinsic viscosity of 100 cm³ /g and asubstitution degree of acetyl group of 3.2;

Sample solution 7: 0.2% aqueous solution of low molecular weight sodiumacetylhyaluronate having a intrinsic viscosity of 90 cm³ /g and asubstitution degree of acetyl group of 3.4;

Sample solution 8: 0.2% aqueous solution of sodium hyaluronate (HA)having a molecular weight of 900,000; and

Sample solution 9: 1% aqueous solution of propylene glycol (PG).

The results thereof is shown in FIG. 6.

As can be seen from this chart, as compared with ion-exchanged water,moisturizing effect is high when sodium hyaluronate or propylene glycol,which is a typical humectant, is added; while sodium acetylhyaluronatehas a moisturizing effect which is further superior to that of sodiumhyaluronate or propylene glycol.

Skin-Softening Effect Pretest

Skin-softening effect measurement test was conducted as mentioned abovewith respect to the following sample solutions:

Sample solution 1: ion-exchanged water;

Sample solution 2: 0.2% aqueous solution of low molecular weight sodiumacetylhyaluronate having a intrinsic viscosity of 120 cm³ /g and asubstitution degree of acetyl group of 3.0;

Sample solution 3: 0.2% aqueous solution of low molecular weight sodiumacetylhyaluronate having a intrinsic viscosity of 110 cm³ /g and asubstitution degree of acetyl group of 3.1;

Sample solution 4: 0.2% aqueous solution of low molecular weight sodiumacetylhyaluronate having a intrinsic viscosity of 100 cm³ /g and asubstitution degree of acetyl group of 3.2;

Sample solution 5: 0.2% aqueous solution of sodium hyaluronate (HA)having a molecular weight of 900,000; and

Sample solution 6: 5% aqueous solution of glycerine (DG).

The results thereof are shown in FIG. 7.

As can be seen from this chart, in the section to which ion-exchangedwater has been applied, until 20 minutes after the application, theelastic modulus ratio decreases due to the moisture infiltrating intothe keratin layer, thereby increasing the softness of the keratin layer.Then, about 30 minutes after the application, due to being elasticresulting from water evaporation, the elastic modulus ratio returns tothe value before the application and, due to the rebound in waterevaporation, further to a value slightly exceeding 1. In the section towhich aqueous sodium hyaluronate has been applied, the recovered valueof softness is slightly lower than that in the section whereion-exchanged water has been applied and there is a slight effect. Itis, however, recovered to about 0.9 in about 50 minutes, thereby beingunderstood that thre is a low stratum corneum softening effect. Bycontrast, unlike the sections where ion-exchanged water and hyaluronicacid have been applied, the section to which the aqueous glycerinesolution has been applied maintains the value of about 0.7, therebyyielding a relatively high softness maintaining effect.

On the other hand, in the section to which low molecular weight sodiumacetylhyaluronate has been applied, the elastic modulus ratio ismaintained at about 0.3 even 120 minutes after the application, therebyyielding a very high stratum corneum softening effect.

Based on these test results, it has been decided that the skin-softeningeffect would be evaluated by the average value of the elastic modulusratio at least 60 minutes after the application in view of the softnessmaintaining effect in the keratin layer.

As can be seen from the foregoing pretests, it is understood thatacetylhyaluronate is much superior to hyaluronic acid or the like interms of thread-forming ability, moisturizing effect, and skin-softeningeffect, for example.

Next, each parameters in the above-mentioned method of manufacturingacetylhyaluronate was changed so as to prepare various kinds ofacetylhyaluronate having various intrinsic viscosity (molecular weight)values and numbers of acetyl group substitution, by which theabove-mentioned effects were inspected.

The results thereof will be shown in the following.

First, in Table 1, various kinds of acetylhyaluronate having respectivesubstitution degrees of acetyl group were prepared with a intrinsicviscosity of about 120 cm³ /g to inspect various effects mentionedabove. The results thereof are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                                  HA   AcHA                                                           ______________________________________                                        Intrinsic viscosity                                                                       1400   125    122  120  118  115  113                             (cm.sup.3 /g)                                                                 Substitution degree of                                                                    0      2.6    2.8  3.0  3.2  3.4  3.6                             acetyl group                                                                  Thread length (cm)                                                                        2.00   0.45   0.40 0.34 0.32 0.31 0.30                            Constant of water                                                                         2.03   2.01   1.91 1.87 1,87 1.90 2.00                            evaporation rate                                                              (g/min)                                                                       Elastic modulus ratio                                                                     0.92   0.89   0.61 0.32 0.26 0.54 0.87                            ______________________________________                                    

As clearly from Table 1, in cases where the intrinsic viscosity is about120 cm³ /g, there was remarkable improvement in thread-forming ability,skin-softening effect, and moisturizing effect when the substitutiondegree of acetyl group is 2.6 or more. However, when the substitutiondegree of acetyl group was 3.6, moisturizing effect and skin-softeningeffect tend to decrease so as to be difficult it to be used for anexternal preparation for skin.

Next, the inventors prepared various kinds of acetylhyaluronate havingrespective intrinsic viscosity values with a substitution degree ofacetyl group at about 3.0 and inspected their effects.

                  TABLE 2                                                         ______________________________________                                                  HA   AcHA                                                           ______________________________________                                        Intrinsic viscosity                                                                       1400   200    160  120  100  80   50                              (cm.sup.3 g/)                                                                 Substitution degree of                                                                    0      2.8    2.9  3.0  3.1  3.0  3.1                             acetyl group                                                                  Thread length (cm)                                                                        2.00   1.03   0.61 0.34 0.21 0.10 0.08                            Constant of water                                                                         2.03   1.99   1.89 1.87 1.85 1.94 2.01                            evaporation rate                                                              (g/min)                                                                       Elastic modulus ratio                                                                     0.92   0.80   0.57 0.32 035  0.61 0.79                            ______________________________________                                    

The above results indicate that, in cases where the substitution degreeof acetyl group is about 3.0, when the intrinsic viscosity exceeds 200cm³ /g, improvement in thread-forming ability becomes insufficient evenby acetylation. On the other hand, when the intrinsic viscosity is lessthan 50 cm³ /g, moisturizing effect and skin-softening effect, forexample, tend to become insufficient.

In the following, more specific examples of the cosmetic preparation inaccordance with the present invention will be explained.

    ______________________________________                                        (1) Dipropylene glycol        5.0                                             (2) Glycerine                 8.0                                             (3) Carboxyvinylpolymer       0.1                                             (4) Triethanolamine           1.0                                             (5) Stearic acid              2.0                                             (6) Sorbitan monooleate       2.0                                             (7) Stearyl alcohol           1.5                                             (8) Vaseline                  4.0                                             (9) Squalane                  5.0                                             (10) Glycerol tri-2-ethylhexanoate                                                                          2.0                                             (11) Ethyl paraben            0.2                                             (12) Perfume                  0.05                                            (13) Acetylhyaluronate        0.1                                             (intrinsic viscosity: 102 cm.sup.3 /g, substitution degree of acetyl          group:                                                                        3.2)                                                                          (14) lon-exchanged water      47.05                                           ______________________________________                                    

A milky lotion was prepared by the conventional method. It was favorablein terms of affinity and wetness according to a panel evaluation.

EXAMPLE 2 Moisturizing Cream

    ______________________________________                                        (1) 1,3-Butyleneglycol        9.5                                             (2) Acetylbyaluronate         0.5                                             (intrinsic viscosity: 72 cm.sup.3 /g, substitution degree of acetyl           group:                                                                        3.0)                                                                          (3) POE(25) cetyl alcohol ether                                                                             3.0                                             (4) Glycerine monostearate    2.0                                             (5) Cetyl alcohol             3.0                                             (6) Solid paraffin            2.0                                             (7) Vaseline                  5.0                                             (8) Squalane                  15.0                                            (9) Butyl paraben             0.2                                             (10) Chondroitin              0.05                                            (11) Perfume                  0.1                                             (12) Ion-exchanged water      59.65                                           ______________________________________                                    

A moisturizing cream was prepared by the conventional method. It wasfavorable in terms of affinity and wetness according to a panelevaluation.

EXAMPLE 3 Massage Cream

    ______________________________________                                        (1) Acetylhyaluronate         1.0                                             (intrinsic viscosity: 180 cm.sup.3 /g substitution degree of acetyl           group:                                                                        3.1)                                                                          (2) POE(20) sorbitan monwmte  2.0                                             (3) Glycerine monostearate    2.5                                             (4) Stearic acid                                                              (5) Potassium hydroxide       0.1                                             (6) Cetyl alcohol             3.0                                             (7) Solid paraffin            5.0                                             (8) Vaseline                  10.0                                            (9) Liquid parraffin          35.0                                            (10) Isopropyl myristate      10.0                                            (11) Butyl paraben            0.2                                             (12) Alanine                  0.05                                            (13) Perfume                  0.1                                             (14) Ion-exchanged water      29.05                                           ______________________________________                                    

A massage cream was prepared by the conventional method. It wasfavorable in terms of affinity and wetness according to a panelevaluation.

EXAMPLE 4 Cleansing Cream

    ______________________________________                                        (1) Stearic acid        12.0                                                  (2) Myristic acid       14.0                                                  (3) Lauric acid         5.0                                                   (4) Jojoba oil          3.0                                                   (5) Potassium hydroxide 5.0                                                   (6) Sorbitol (70% aqueous sorbitol solution)                                                          20.0                                                  (7) Acetylhyaluronate   2.0                                                   (intrinsic viscisity: 148 cm.sup.3 /g, substitution degree of                 acetyl group: 3.4)                                                            (8) 1,3-Butyleneglycol  13.0                                                  (9) POE(20) glycerol monostearate                                                                     2.0                                                   (10) Acylmethyltaurine  4.0                                                   (11) Glycine            0.01                                                  (12) Chelating agent    appropriate amount                                    (13) Perfume            appropriate amount                                    (14) Ion-exchanged water                                                                              20.0                                                  ______________________________________                                    

<Method of Preparation>

Ingredients (1) to (4), (6) to (8), and (11) were dissolved by heatingand the mixture was maintained at 70° C. Ingredient (5) was dissolved iningredient (14) and the above-mentioned mixture was added thereto whilebeing stirred. After a sufficient neutralization reaction, ingredients(9) and (10) were added thereto and then ingredients (12) and (13) wereadded thereto. After deaeration and cooling, a cleansing foam wasobtained.

It was favorable in terms of affinity and wetness according to a panelevaluation.

EXAMPLE 5 Hair Tonic

    ______________________________________                                        (1) Ethanol             75.0                                                  (2) Ion-exchanged water 23.2                                                  (3) Percutaneous absorption accelerator                                                               appropriate amount                                    (4) Solubilizer         appropriate amount                                    (5) pH adjustor         appropriate amount                                    (6) Perfume             appropriate amount                                    (7) Acetylhyaluronate    0.1                                                  (intrinsic viscosity: 120 cm.sup.3 /g, substitution degree of acetyl          group: 3.0)                                                                   ______________________________________                                    

A hair tonic was prepared by the conventional method. It yielded nosliminess, a favorable affinity, and a refreshing feel of use accordingto a panel evaluation.

EXAMPLE 6 Softening Lotion

    ______________________________________                                        (1) Ion-exchanged water 90.2                                                  (2) Acetylhyaluronate   0.1                                                   (intrinsic viscosity: 120 cm.sup.3 /g, substitution degree                    of acetyl group: 3.0)                                                         (3) 1,3-Butyleneglycol  5.0                                                   (4) Glycerine           4.0                                                   (5) Antiseptic          appropriate amount                                    ______________________________________                                    

A softening lotion was prepared by the conventional method. It yieldedno sliminess, a favorable affinity, and a refreshing feel of useaccording to a panel evaluation.

As explained in the foregoing, since low molecular weightacetylhyaluronate in accordance with the present invention has aspecific molecular weight and substitution degree of acetyl group, itcan exhibit excellent skin-softening effect and moisturizing effect,while appropriately suppressing the thread-forming ability inherent inhyaluronic acid.

We claim:
 1. Acetylhyaluronate having the followingcharacteristics:intrinsic viscosity: 50 to 200 cm³ /g substitutiondegree of acetyl group: 2.6 to 3.6 (excluding N-acetyl group).
 2. Askin-softening composition mainly composed of acetylhyaluronateaccording to claim
 1. 3. A method of manufacturing acetylhyaluronatecomprising the steps of suspending hyaluronic acid powder in an aceticanhydride solvent and then adding concentrated sulfuric acid thereto toeffect acetylation.
 4. A method of manufacturing acetylhyaluronateaccording to claim 3, wherein acetic acid is mixed into the aceticanhydride solvent with a mixing ratio of acetic acid:acetic anhydride at1:4 to 1:1.
 5. A method of manufacturing acetylhyaluronate according toclaim 3, wherein acetic acid is mixed into the acetic anhydride solventwith a mixing ratio of acetic acid:acetic anhydride at 2:1 to 4:1.
 6. Amethod according to claim 3, wherein an amount of concentrated sulfuricacid added is 2 to 7% by volume with respect to the solvent.
 7. A methodof purifying acetylhyaluronate comprising the steps of adding rawacetylhyaluronate to an aqueous acetone solution, adding and dissolvingsodium lactate therein, and then adding acetone thereto to precipitateand obtain said purified acetylhyaluronate.
 8. A method according toclaim 7, wherein an amount of sodium lactate added is 1 to 3% by weightwith respect to the aqueous acetone solution.
 9. A method of purifyingacetylhyaluronate, wherein the method according to claim 7 is applied toraw acetylhyaluronate obtained by a method of manufacturing comprisingthe steps of suspending hyaluronic acid powder in an acetic anhydridesolvent and then adding concentrated sulfuric acid thereto to effectacetylation.